The following is an explanation of the background of the present invention together with a listing which, in the opinion of the Applicants, sets forth the closest prior art of which the Applicants are aware. A concise explanation of the relevance of the more important items is included.
Viral hepatitis has assumed significant world-wide epidemic proportions. It is estimated that there are two hundred million carriers of hepatitis B virus ("HBV") worldwide. The development of a conventional vaccine has been hampered by the inability to grow hepatitis B virus in tissue culture. As a result, it has been necessary to produce hepatitis B subunit particle vaccines by isolating and purifying the 22 nm lipoprotein particles composed of hepatitis B surface antigen ("HBsAg") from plasmas of asymptomatic human carriers. However, such formalin- or heat-inactivated vaccines have the disadvantages of substantial expense and limited supply. In addition, such a source presents potential hazards in view of unknown factors that may be present in the plasma. Also, as high-risk populations are immunized, sources of plasma containing large quantities of HBsAg will become scarce.
Chemically synthesized vaccines to replace viral vaccines offer the advantage of precise biochemical characterization, exclusion of genetic material of viral origin, exclusion of host- or donor-derived substances, consistent potency, and the like. Ideally, such synthetic vaccines do not have irrelevant microbial antigenic determinants, proteins or other materials that might otherwise contaminate the essential immunogen and cause unwanted side effects.
The possibility of a synthetic peptide vaccine for HBV has been suggested in the past. The following is a list of references relating to such vaccines or processes with respect thereto and which will be referred to herein by the respective reference numbers:
1. McAuliffe, V. J., Purcell, R. H. & Gerin, J. L. Rev. Infect. Dis. 2, 470-492 (1980). PA0 2. Rao, K. R. & Vyas, G. N. Nature New Biol. 241, 240-241 (1973). PA0 3. Melnick, J. L., Dreesman, G. R. & Hollinger, F. B. J. Infect. Dis. 133, 210-229 (1976). PA0 4. Anderer, F. A. Biochim. Biophys. Acta 71, 246-248 (1963). PA0 5. Fearney, F. J., Leung, C. Y., Young, J. D. & Benjamini, E. Biochim. biophys. Acta 243, 509-514 (1971). PA0 6. Langbeheim, H., Arnon, R. & Sela, M. Proc. Natn. Acad. Sci. U.S.A. 73, 4636-4640 (1976). PA0 7. Tiollais, P., Charnay, P. & Vyas, G. N. Science 213, 406-411 (1981). PA0 8a. Peterson, D. L. J. Biol. Chem. 256, 6975-6983 (1981). 8b. Gavilaives, F., Gonzalez-Ros, F.M. & Peterson, D.L. J. Biol. Chem 257, 7770 (1982). PA0 8c. Peterson, D. L. In Viral Hepatitis (Eds. W. Szmuness, H. J. Alter and J. E. Maynard). Franklin Institute Press, Philadelphia, p. 707 (1982). PA0 9a. Lerner, R. A. et al Proc. Natn. Acad. Sci. U.S.A. 78, 3403-3407 (1981). PA0 9b. Prince, A. M., Ikram, H. & Hopp, T. P. Proc. Natl. Acad. Sci. U.S.A. 79, 579 (1982). PA0 9c. Bhatnagar, P. R., Papas, E., Blum, H. E., Milich, D. R., Nitecki, D., Karels, M. J. & Vyas, G. N. Proc. Natl. Acad. Sci. U.S.A. 79, 4400 (1982). PA0 10. Chou, P. Y. & Fasman, G. D. Adv. Enzym. 47, 45-148 (1978). PA0 11. Bull, H. B. & Breese, K. Archs Biochem. Biophys. 161, 665-670 (1975). PA0 12. Atassi, M. Z. Immunochemistry 12, 423-438 (1975). PA0 13. Merrifield, R. B. Adv. Enzym. 32, 221-296 (1969). PA0 14. Edelstein, M. S., McNair, D. S. & Sparrow, J. T. in Peptides:Synthesis, Structure, Function (eds Rich, D. H. & Gross, E.) (Pierce Chemical Co., Rockford, pp. 217-220), 1981. PA0 15. Sparrow, J. T. J. Org. Chem. 41, 1350-1353 (1976). PA0 16. Atherton, E., Woolley, V. & Sheppard, R. C. JCS Chem. Commun., 970 (1981). PA0 17. Mao, S. J. T., Sparrow, J. T., Gilliam, E. B., Gotto, A. M. & Jackson, R. L. Biochemistry 16, 4150-4156 (1977). PA0 18. Felix, A. M., Jiminez, M. H., Wang, C. T. & Meienhofer, J. Int. J. Peptide Protein Res. 15, 342-354 (1980). PA0 19. Dreesman, G. R., Hollinger, F. B., Sanchez, Y., Oefinger, P. & Melnick, J. L. Infect. Immun. 32, 62-67 (1981). PA0 20. Sanchez, Y. et at Infect. Immun. 30, 728-733 (1980). PA0 21. Dixon, W. J. & Brown, M. B. BMDP-79 (University of California Press, Berkeley, 1979). PA0 22. Hollinger, F. B., Adam, E., Heiberg, D. & Melnick, J. L. in Viral Hepatitis (eds. Szmuness, W., Alter, H., & Maynard, J.) (Franklin Institute Press, Philadelphia, pp. 451-466, 1982). PA0 23. Fudenberg, H. H. & Kunkel, H. G. J. Exp. Med. 106, 689-702 (1957). PA0 24. Vyas, G. N. in Hepatitis B Vaccine (eds. Maupas, P. & Guesry, P.) (Elsevier, Amsterdam, 1981). PA0 25. Hopp, T. P. & Woods, K. R. Proc. Natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981). PA0 26. Zuckerman, A. J. New Scient. 88, 167 (1980). PA0 27. Skelly et al Nature 290, 51 (1981). PA0 28. Hopp, T. P. Molec. Immun. 18, 869 (1981). PA0 29. Charnay, P., Pourcel, C., Louise, A., Fritsch, A. and Tiollais, P. Proc. Natl. Acad. Sci. U.S.A. 76, 2222 (1979). PA0 30. Valenzuela, P., Gray, P., Quiroga, M., Zaldivar, J., Goodman, A. M. and Rutter, W. J. Nature 280, 815 (1979). PA0 31. Pasek, M., Golo, T., Gilbert W., Zink, B., Schaller, H., McKay, P., Leadbetter, G. and Murray, K. Nature 282, 575 (1979). PA0 32. Allison, A. C., Buckland, F. E. and Andrewes, C. H. Virology 17, 171 (1962). PA0 33. Carver, D. H. and Seto, D. S. Y. J. Virol. 2, 1482 (1968). PA0 34. Hare, J. D. and Chan, J. C. Virology 34, 481 (1968). PA0 35. Sukeno, N., Shirachi, R., Yamaguchi, J. and Ishida, N. J. Virol. 9, 182 (1972). PA0 36. Vyas, G. N., Rao, K. R. and Ibrahim, A. B. Science 178, 1300 (1972). PA0 37. Dreesman, G. R., Hollinger, F. B., McCombs, R. M. and Melnick, J. L. J. Gen. Virol. 19, 129 (1973). PA0 38. Kohler, G. and Milstein C.: Continuous culture of fused cells secreting antibody of predefined specificity. PA0 39. Oudin, J., and Michel, M. 1963. Une novelle formed' allotypie des globulines y du serum de lapin aparement lie'e a le jonction et a la specificite anticoyss. C. R. Seanc. Soc., Paris 257:805. PA0 40. Hollinger, F. B., Dreesman, G. R., Sanchez, Y., abral, G. A. and Melnick, J. L. Experimental hepatitis B polypeptide vaccine in chimpanzees. In Viral Hepatitis (eds. G. N. Vyas, S. N. Cohen and R. Schmid). Franklin Institute Press, Philadelphia, pp. 557-567, 1978.
Nature (London) 256:495-497 (1975).
Reference 1 relates to formalin-inactivated hepatitis B virus vaccines that have been produced in several laboratories. The sole source of material for these vaccines has been 22-nm lipoprotein particles composed of HBsAg and derived from plasma of persons chronically infected with HBV.
The possibility of a synthetic peptide vaccine for HBV was suggested in References 2 and 3 following studies carried out with tobacco mosaic virus (References 4 and 5) and MS-2 coliphage (Reference 6). This possibility became a reality when the amino acid sequence for HBsAg was deduced from the nucleotide sequence of the cloned HBV genome as reviewed in Reference 7. Reference 8 discloses that a portion of the major polypeptide derived from HBsAg, with a calculated molecular weight of 25,000 (P25), has been sequenced.
Hepatitis B polypeptide vaccines containing hepatitis B-specific antigenic determinants associated with a nonglycosylated polypeptide with a molecular weight in the range 22,000-24,000 and a glycosylated polypeptide with a molecular weight in the range 26,000-29,000 have been prepared and tested for safety, immunogenicity and protective efficicacy in susceptible chimpanzees. (References 19, 26, 27 and 40). The non-glycosylated polypeptide and the glycosylated polypeptide referred to herein have molecular weights of 25,000 (P25) and 30,000 (GP30), respectively, as determined by their amino acid sequence deduced from the sequence of the cloned hepatitis B virus DNA genome. (Reference 7)
Previous studies have demonstrated that sulfhydryl groups and/or disulfide bonds play an important role in the tertiary structure of a number of animal viruses since biological activities such as infectivity and hemagglutination are destroyed by treatment with either alkylating (sulfhydryl binding) or reducing reagents (References 32-34). The differential effect of these different reagents is illustrated by the fact that a reducing agent such as dithiothreitol destroys the infectivity of many enteroviruses, but these same viruses are unaffected by treatment with sulfhydryl binding reagents (References 32, 33). More specifically, it has been shown that the antigenic determinants (epitopes) associated with HBsAg are conformation-dependent. This was demonstrated in that reduction of the disulfide bonds contained in HBsAg and subsequent alkylation of the free thiol groups destroyed both the antigenic and the immunogenic activities associated with HBsAg (References 35-37).
Reference 28 describes a computerized analysis of the amino acid sequences of the HBsAg protein to predict an antigenic site determinant. An amino acid sequence of residues 138-149 was synthesized and examined for its ability to bind antibodies to a mixture of the ad and ay subtypes of HBsAg. The peptide bound 9% of the antibodies.
As reported in Reference 9a, thirteen peptides were chemically synthesized corresponding to amino acid sequences predicted from the nucleotide sequence for HBsAg. Seven out of the thirteen synthetic peptides elicited an anti-peptide response in rabbits inoculated with three or four doses of a series of peptides, each containing 14-15 amino acid residues, but only after covalent linkage of the peptides to a carrier protein. Activity also was found after multiple injections of a peptide containing 34 amino acids. In Reference 9b a synthetic peptide containing amino acid residues 138-149 of P25 was prepared. This peptide was reported to contain the a group reactivity as well as d subgroup reactivity. When this peptide was conjugated to human erythrocytes and injected into mice, it induced the formation of anti-HBs with or without the use of Freund's adjuvant. The investigation in Reference 9c prepared seven linear peptide analogues of HbsAg: 122-137, 128-134, 139-147, 139-158, 140-158, 145-158, and 150-158. For experimental immunization of rabbits the synthetic peptides were coupled to keyhole limpet hemocyanin. The investigators studied the antigenicity of each peptide analogue by serologic neutralization of human antibodies specific for the a determinant of HBsAg. Analogues 139-147, 139-158, and 140-158 showed antigenicity as well as induction of anti-HBsAg. The rabbit antibodies were inhibited with each of the three peptide analogues and all serotypes of natural HBsAg, having only the a determinant in common. They reported that a linear form of peptide 122-137 and of peptide 128-134 covalent linked to a protein carrier failed to elicit production of anti-HBs in rabbits. (Reference 9c).
Two patent applications pertaining to the production of synthetic peptides with application to viral vaccines (with specific reference to HBsAg) have been noted. The first, by R. A. Lerner et al. (European Patent Application No. 044,710 filed July 16, 1981) describes the production of multiple synthetic peptides containing different amino acid sequences associated with the native P25 polypeptide derived from HBsAg. The Lerner et al. application differs from the present invention in two distinct areas. First, the Lerner et al. peptides are uniformly linear, and Lerner et al. made no attempt to produce tertiary conformation as described herein. Secondly, there was no attempt to purify the synthetic peptides before coupling to the protein carrier. Thirdly, Lerner et al. produced antibody to a peptide only after conjugation to a carrier. The antibody produced reacted with native HBsAg but no attempt was made to determine whether the linear peptides reacted with antibodies produced in human beings following a natural hepatitis B viral infection. As explained herein, the reaction of our circular peptide with human hepatitis B antibodies demonstrates the importance of cyclization of synthetic HBsAg peptides.
The second patent application by T. P. Hopp (European Patent Application No. 056,249 filed Jan. 8, 1982) describes the use of a computer program analysis modified to predict the most hydrophilic region of viral and bacterial polypeptides. He has applied this to the synthesis of a peptide containing 6 amino acid residues corresponding to amino acids 141-147 of the P25 protein of HBsAg. Similar to the teachings of Lerner et al. mentioned above, Hopp's peptides are uniformly linear and no attempt has been made to construct the tertiary structure associated with the conformation dependent antigenic determinants of HBsAg. Neither Lerner et al. nor Hopp mentions cyclization of their peptides by formation of intrachain or interchain disulfide bonds as in the present invention.
None of the above references teaches or suggests cyclic peptides containing disulfide bonds in a hydrophilic region of the major viral polypeptide which, as will be described hereafter, elicits an antibody response in mice after a single injection without linkage to a protein carrier. The composition according to the present invention contains a determinant that elicits the production of antibody of similar specificity to that produced by immunization with the SDS-denatured linear virus P25 polypeptide described in References 19 and 40. While the synthetic peptides disclosed in Reference 9a have several amino acids (138-146) in common with the compositions according to the present invention and there are four overlapping carboxyl amino acids of the peptides, there are no other substantial similarities between the teachings of References 9a,9b and 9c and the present invention.